User:Judy Voet/Lysozyme

 Lysozyme was the first enzyme whose X-ray structure was determined. This scene shows Hen Egg White (HEW) lysozyme  containing a trisaccharide of N-acetylglucosamine (NAG) bound to a cleft in the enzyme. David Phillips, who determined the structure in 1965, saw that the cleft was large enough to fit three more saccharide units. He therefore built a model extending the trisaccharide to a hexasaccharide that fits into the cleft, labeling the sugar subsites A-F. Alternately click on trisaccharide and hexasaccharide to turn the modeled portion of the hexasaccharide on and off. The interesting thing about the model was that the only way that the hexasaccharide would fit into the cleft was if the 4th saccharide (in subsite D) was strained into a half-chair conformation. This conformation is what would be necessary for the formation of an oxocarbenium ion (oxionium ion). When the model was studied, Glu 35 was found to be in an ideal location to act as a general acid catalyst, 3.34 Angstroms from the bridging oxygen between the 4th and 5th saccharide units. Asp 52 appeared to be too far away (2.69 angstroms) in the static lysozyme structure to have formed a covalent bond with C1 of the half-chair model in the D site, and no covalent intermediate had ever been detected, so Phillips proposed that it acted as an electrostatic stabilizer of the oxonium ion (referred to as The Phillips Mechanism).

 Then, in 2001, Stephen Withers published 1H6M, in which Glu 35 he had mutated to Gln to remove the general acid catalyst. The substrate contained NAG-2-fluoro-glucosyl fluoride (NAG2FGlcF). The fluoro group on C-1 does not require acid catalysis to be a good leaving group, and the remaining saccharide, in the absence of the acid necessary to catalyse the second step of the reaction, was demonstrated to form a  covalent intermediate. In this superposition of the half chair model with 1HEW (greens) and the covalent intermediate in 1H6M (blues), note  the relatively small motions of Asp 52 and C1 of the sugar ring in going from the model to the covalent intermediate. to observe the motion from the half-chair to the covalent intermediate just toggle between the two green links.

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Lysozyme

Retaining Glycoside Hydrolases